Molecular dynamics simulations study of influence of Tyr422Ala mutation on transcriptional enhancer activation domain 4 (TEAD4) and transcription co-activators complexes.

Transcriptional enhancer activation domain (TEAD) proteins are the downstream transcriptional factor of the Hippo pathway. The transcription co-activators Yes-associated protein (YAP) and its paralog transcription co-activators with PDZ-binding motif (TAZ), binding to TEAD to promote transcription of genes in cell proliferation and anti-apoptosis, are key effectors of the Hippo pathway. TEAD4, one member of TEAD proteins, is specifically required in embryo implantation. The recently reported crystal structure of TEAD4-TAZ complex (PDB Code 5GN0) in mouse reveals that the interactions between the two helices of YAP/TAZ and TEAD4 are highly conserved. Point mutation of the residue Tyr422 of TEAD4 protein would disrupt the relevant hydrogen bond and even abolish the interaction. However, detailed information affected by the mutation at the atom level are still unrevealed. Molecular dynamics (MD) simulations and the molecular mechanics/Generalized-Born surface area (MM/GBSA) free energy calculations were used to explore the effects of mutation Tyr422Ala on the structural flexibility and conformational dynamics. The non-polar interactions play an indispensable role in the binding process of TEAD4 and YAP/TAZ. The helices α1 and α2 of YAP/TAZ provide a primary function to anchor YAP/TAZ well bound to TEAD4. The mutation Tyr422Ala disrupts the hydrogen-bonding network but do not obviously influence the secondary structure stability of TEAD4. The binding conformation of YAP/TAZ distorted by decreased non-polar interaction and the lost hydrogen bonds would lead to reduced interaction activity. The present study would provide important insights into the structure-function relationships of TEAD protein and give a new explanation for the affinity of YAP/TAZ with TEAD.

The influence of residue in the position of 116 on the inhibitory potency of TH588 for MTH1.

As one of the first-in-class inhibitor, TH588 was found to be efficient in the suppression of MutT homolog1 (MTH1). A recent work shows that the inhibitory potency of TH588 against human MTH1 (hsMTH1) is approximately 20-fold over that of mouse MTH1 (mmMTH1) and identifies residue in position 116 in MTH1 has an important contribution to TH588 affinity. But the effect of residue Leu or Met in position 116 on the binding affinity remains unclear. In this study, molecular dynamics (MD) simulations and free energy calculations were used to elucidate the mechanism about the effect of residue 116 to the different inhibitory potency of TH588 against MTH1. The binding free energy of TH588 in M116 complexes predicated by the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) is much lower than that in L116 complexes, which is consistent with the experiment results. The analysis of the individual energy terms suggests that the non-polar interactions are important for distinguishing the binding of TH588. The MD results show that the Leu116 disrupts the interactions between Asn33 and TH588, thus induces the conformational changes of Asn33 as well as TH588. The altered interactions between TH588 and mmMTH1 change the flexibility of TH588, which could induce the remarkable conformational fluctuation of mmMTH1. The conformations of the two loops covering the binding pocket have obvious influence on the opening or closure of the active site. The more open binding site may explain the lower inhibitor potency of TH588 against mmMTH1 than hsMTH1. Our results provide mechanistic insight into the effect of different residue Leu or Met in position 116 on the binding affinity of TH588 for MTH1, which is expected to contribute to the further rational design of more potent inhibitors.

Molecular basis of the recognition of FMN by a HAD phosphatase TON_0338.

The haloalkaloic acid dehalogenase (HAD) phosphatase from Thermococcus onnurineus NA1 (TON_0338), has phosphatase activity the flavin mono-nucleotide (FMN). The molecular origin and structural motifs for the activity deficiency of double-tryptophan mutant have not been rationalized at atomic resolution. Molecular dynamics (MD) simulations and the molecular mechanics/Generalized-Born surface area (MM/GBSA) free energy calculations were used to explore the effects of mutations on the changes in both structural flexibility and conformational dynamics. The non-polar solvation energy plays an indispensable role in the binding process of TON_0338 and FMN. The tryptophan sandwich structure provides a primary function to anchor FMN and keeps FMN well bound to TON_0338. The double-tryptophan mutation has influences on the secondary structures of TON_0338 and changes the conformation, which would lead to reduced activity of W58A/W61A-FMN binding. The present study provides important insights into the structure-function relationships of TON_0338 protein, which could contribute to further understanding about the HAD phosphatases.

Molecular dynamics simulations studies and free energy analysis on inhibitors of MDM2-p53 interaction.

The oncoprotein MDM2 (murine double minute 2) negatively regulates the activity and stability of tumor suppressor p53. Inactivation of the MDM2-p53 interaction by potent inhibitors offers new possibilities for anticancer therapy. Molecular dynamics (MD) simulations were performed on three inhibitors-MDM2 complexes to investigate the stability and structural transitions. Simulations show that the backbone of MDM2 maintains stable during the whole time. However, slightly structural changes of inhibitors and MDM2 are observed. Furthermore, the molecular mechanics generalized Born surface area (MM-GBSA) approach was introduced to analyze the interactions between inhibitors and MDM2. The results show that binding of inhibitor pDIQ to MDM2 is significantly stronger than that of pMI and pDI to MDM2. The side chains of residues have more contribution than backbone of residues in energy decomposition. The structure-affinity analyses show that L54, I61, M62, Y67, Q72, H73 and V93 produce important interaction energy with inhibitors. The residue W/Y22' is also very important to the interaction between the inhibitors and MDM2. The three-dimensional structures at different times indicate that the mobility of Y100 influences on the binding of inhibitors to MDM2, and its change has important role in conformations of inhibitors and MDM2.

Structural and dynamic basis of human cytochrome P450 7B1: a survey of substrate selectivity and major active site access channels.

Cytochrome P450 (CYP) 7B1 is a steroid cytochrome P450 7α-hydroxylase that has been linked directly with bile salt synthesis and hereditary spastic paraplegia type 5 (SPG5). The enzyme provides the primary metabolic route for neurosteroids dehydroepiandrosterone (DHEA), cholesterol derivatives 25-hydroxycholesterol (25-HOChol), and other steroids such as 5α-androstane-3ß,17ß-diol (anediol), and 5α-androstene-3ß,17ß-diol (enediol). A series of investigations including homology modeling, molecular dynamics (MD), and automatic docking, combined with the results of previous experimental site-directed mutagenesis studies and access channels analysis, have identified the structural features relevant to the substrate selectivity of CYP7B1. The results clearly identify the dominant access channels and critical residues responsible for ligand binding. Both binding free energy analysis and total interaction energy analysis are consistent with the experimental conclusion that 25-HOChol is the best substrate. According to 20 ns MD simulations, the Phe cluster residues that lie above the active site, particularly Phe489, are proposed to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. The investigation of CYP7B1-substrate binding modes provides detailed insights into the poorly understood structural features of human CYP7B1 at the atomic level, and will be valuable information for drug development and protein engineering.

Analysis of clinically relevant substrates of CYP2B6 enzyme by computational methods.

Mounting evidence thus far indicates that human cytochrome P450 2B6 (CYP2B6), an enzyme expressed at a relatively low level functionally, is primarily responsible for the metabolism of several clinically relevant drugs, including propofol, efavirenz, bupropion, mephobarbital, and the propofol analog 2,6-di-sec-butyl phenol. We used molecular dynamics and molecular docking methods to predict such interactions and to compare with experimentally measured metabolisms. Insight II and Discover Studio 2.5 were used to carry out the docking of these substrates into CYP2B6 to explore the critical residues and interaction energies of the complexes. Phe297, Glu301, Thr302 and Val367 were identified as major drug-binding residues, which is consistent with previous data on site-directed mutagenesis, crystallography structure, and from modeling and docking studies. In addition, our docking results suggest that nonpolar amino acid clusters and heme also participate in binding to mediate drug oxidative metabolism. The binding modes of the five clinically relevant substrates mentioned above for metabolism on CYP2B6 are presented.